Volume 14, Issue 5, Pages 583-590 (May 2001) GADD45γ Mediates the Activation of the p38 and JNK MAP Kinase Pathways and Cytokine Production in Effector TH1 Cells  Binfeng Lu, Hong Yu, Chi-wing Chow, Baiyong Li, Wei-ping Zheng, Roger J Davis, Richard A Flavell  Immunity  Volume 14, Issue 5, Pages 583-590 (May 2001) DOI: 10.1016/S1074-7613(01)00141-8

Figure 1 GADD45γ Is Highly Induced in TH1 Cells Naive (CD62L+CD44−) CD4+ T cells (lane 1) were isolated from 8-week-old C57/B6 mice. These cells were then cultured on α-CD3 (10 μg/ml) and α-CD28 (5 μg/ml) coated plates. Cells were cultured under TH1, TH2, or no polarizing conditions for 48 and 72 hr Immunity 2001 14, 583-590DOI: (10.1016/S1074-7613(01)00141-8)

Figure 2 Generation of GADD45γ−/− Mice (A) The targeting strategy is illustrated schematically. (B) Southern analysis of genomic DNA prepared from tails of GADD45γ+/+, GADD45γ−/−, and GADD45γ+/− mice. (C) Northern analysis of total RNA prepared from CD4+ T cells that were isolated from either GADD45γ+/+ or GADD45γ−/− mice and cultured under TH1 conditions for 3 days. The probe was prepared using a full-length GADD45γ cDNA Immunity 2001 14, 583-590DOI: (10.1016/S1074-7613(01)00141-8)

Figure 3 Proliferation of TH1 Effector Cells Is Not Affected by Mutation of GADD45γ Naive (CD62L+CD44−) CD4+ T cells from either GADD45γ+/+ or GADD45γ−/− mice are cultured under TH1 conditions for 4 days. These cells were harvested and washed three times. Cells were stimulated with plate-bound α-CD3 at the indicated concentration for 24 hr. Then, cells were pulsed with [3H]thymidine for another 16 hr. Proliferation was assayed by determining the incorporation of [3H]thymidine Immunity 2001 14, 583-590DOI: (10.1016/S1074-7613(01)00141-8)

Figure 4 JNK and p38 MAP Kinase Activation in TH1 Effector Cells Is Dependent on GADD45γ (A) Naive (CD62L+CD44−) CD4+ T cells from either GADD45γ+/+ or GADD45γ−/− mice were cultured under TH1 conditions for 5 days. These cells were harvested and washed three times. Cells were stimulated with plate-bound α-CD3 (10 μg/ml) or α-CD3 (10 μg/ml) and α-CD28 (5 μg/ml) for 1 hr. The activated forms of p38 and ERK were detected by immunoblot analysis using phospho-specific antibodies. (B) Naive (CD62L+CD44−) CD4+ T cells from either GADD45γ+/+ or GADD45γ−/− mice were cultured under TH1 conditions for 5 days. These cells were harvested and washed three times and were stimulated with plate-bound α-CD3 (10 μg/ml) for 1 hr. JNK and p38 MAP kinase activity was measured using the substrates c-Jun and ATF2, respectively. (C) Naive (CD62L+CD44−) CD4+ T cells from either GADD45γ+/+ or GADD45γ−/− mice were cultured under TH1 conditions for 4 days. These cells were harvested and washed three times. The cells were stimulated with plate-bound α-CD3 for 15 min. The activated form of p38 was detected by immunoblot analysis using phospho-specific antibodies. (D) Naive (CD62L+CD44−) CD4+ T cells from either GADD45γ+/+ or GADD45γ−/− mice were cultured under the nonpolarizing condition for 4 days. These cells were harvested and washed three times. The cells were stimulated with plate-bound α-CD3 for 15 min. The activated form of p38 was detected by immunoblot analysis using phospho-specific antibodies Immunity 2001 14, 583-590DOI: (10.1016/S1074-7613(01)00141-8)

Figure 5 GADD45γ Deficiency Causes Reduction of Activation-Induced Cell Death Naive (CD62L+CD44−) CD4+ T cells from either GADD45γ+/+ or GADD45γ−/− mice were cultured under the TH1 condition for 5 days. These cells were harvested and washed three times and were stimulated with plate-bound α-CD3 for 8 hr (A) and 24 hr (B). Then, cells were stained with Annexin V and propidium iodide and were analyzed by FACS. Live cells are Annexin V and propidium iodide double-negative cells. The results were the average of three independent experiments, and each was from a different pair of GADD45γ+/+ or GADD45γ−/− mice. Error bars are based on the standard deviation of the data set. TUNEL assay was also carried out, and live cells and TUNEL-positive cells were counted, and the ratio was an average from three fields. The result is representative of three independent experiments. Error bars are based on the standard deviation of the data set Immunity 2001 14, 583-590DOI: (10.1016/S1074-7613(01)00141-8)

Figure 6 GADD45γ Deficiency Compromises IFN-γ Production by TH1 Cells Naive (CD62L+CD44−) CD4+ T cells from either GADD45γ+/+ or GADD45γ−/− mice were cultured under TH1 and TH2 conditions with α-CD3 and α-CD28 antibodies for 5 days. These cells were harvested and washed three times and were stimulated with plate-bound α-CD3 (5 μg/ml) overnight. (A) Enzyme-linked immunosorbent assay (ELISA) analysis of IL-4 production by GADD45γ+/+ and GADD45γ−/− TH2 cells. The data are representative of four independent experiments. (B) IFN-γ production by GADD45γ+/+ and GADD45γ−/− TH1 cells was measured by ELISA. The data are representative of four independent experiments. (C) RNase protection assay (RPA) of IFN-γ mRNA in GADD45γ+/+ and GADD45γ−/− TH1 cells. (D) Naive (CD62L+CD44+) CD4+ T cells from either GADD45γ+/+ or GADD45γ−/− mice were cultured under TH1 conditions with APC and ConA for 4 days. These cells were harvested, washed three times, and were stimulated with plate-bound α-CD3 (5 μg/ml) overnight. IFN-γ production by GADD45γ+/+ and GADD45γ−/− TH1 cells was measured by ELISA. The data are representative of three independent experiments Immunity 2001 14, 583-590DOI: (10.1016/S1074-7613(01)00141-8)

Figure 7 GADD45γ Deficiency Alters Contact Hypersensitivity in Mice Four 8-week-old female wild-type and four 8-week-old female GADD45γ−/− mice were used in the CHS, as described in Experimental Procedures. The statistical significance of differences between the means was determined by applying a Student's t test. Error bars are based on the standard deviation of the data set Immunity 2001 14, 583-590DOI: (10.1016/S1074-7613(01)00141-8)